A cascade of molecular events is triggered in mammalian cells upon exposure to ionizing radiation (IR). These events include DNA damage recognition, damage signaling, DNA repair, and cell cycle control. Although a number of genes and gene products involved in IR responses have been identified, it is not clear how these processes are coordinated. The purpose of this proposal is to understand the inter-relationship among three major DNA damage sensing and signaling proteins: DNA-PK (DNA- dependent protein kinase), ATM (Ataxia Telangiectasia Mutated), and Nbs1 (the gene product mutated in Nijmegen breakage syndrome). We hypothesize that these DNA damage sensing and signaling proteins are coordinated in a temporal and/or spatial fashion in response to DNA damage in mammalian cells. To test this hypothesis, we plan to examine the functional relationship(s) among these proteins in primary mouse embryonic fibroblasts (MEFs) and cell lines derived from transgenic mice that are single or double knockouts (KO) for various combinations of these three proteins. We have generated in the lab or have acquired mice that are knockouts for Ku70, Ku80, DNA-PKcs, Ku80/DNA-PKcs, and ATM. To avoid embryonic and somatic lethality for Nbs1 knockout and ATM/DNA-PKcs double knockout, we plan to generate conditional knockouts for DNA-PKcs and Nbs1 using a novel one-step Tet-Off/CreLoxP strategy designed in our lab. Using these knockout and/or conditional knockout mice, we plan to elucidate 1) the role of the Ku component of DNA-PK in the modulation of ATM kinase activation by DNA damage, 2) the individual and overlapping roles of ATM and DNA-PK, and 3) the role of Nbs1 in the sensing, signaling and repair of DNA breaks. Preliminary results obtained from in vitro as well as from in vivo studies on single and double knockout mice have provided us with insights into these mechanisms. We propose to follow up these leads to obtain a clearer picture of the coordinating mechanisms between DNA-PK, ATM, and Nbs1.